Method of Preparing Nucleic Acids for Detection

ABSTRACT

A method is provided for preparing a test sample for detecting a predetermined target nucleic acid. The method includes the steps of providing a test probe comprising an oligonucleotide attached to a nanoparticle and providing a hybridization unit containing the test sample and the test probe, wherein said hybridization unit further includes a target sample substrate and a distribution manifold coupled to a first side of the substrate. The method further includes the steps of clamping a processing fluids manifold to the distribution manifold of the hybridization unit, denaturing the test sample and preparing the test sample for detecting the predetermined target nucleic acid by pumping a plurality of processing fluids between the processing fluids source manifold and distribution manifold to hybridize the test probe and predetermined target nucleic acid to the target sample substrate, to wash the hybridized sample and to amplify a detectable parameter of the hybridized sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/703,368, filed on Nov. 7, 2003, now U.S. Pat. No. 7,396,677, thedisclosure of which is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of the invention relates to biological testing and moreparticularly to detecting nucleic acids.

2. Description of the Related Art

Methods of detecting nucleic acids are generally known. In fact, thereare a number of methods available for detecting specific nucleic acidsequences.

Known methods include those based upon electrophoresis, polymerase chainreaction (PCR) processes, various hybridization techniques, and a numberof other techniques. While these methods are effective, they are alltime consuming, costly and subject to significant human error.

For example, one manufacturer makes a microfluidics system thathybridizes a sample to a chip followed by staining of the chip. Thehybridization process takes approximately 12 hours. Staining takesapproximately 1.5 hours to complete.

Another supplier provides a system that relies upon a single nucleotidepolymorphism (SNP) technique. This system uses a microchip forperforming multiple assays. Probes are added to a cartridge and theparticles move based on charge in an electric field. A detection systemmay be used for analyzing the cartridges after hybridization with thesample DNA.

Still another supplier provides a device called a LightCycler thatcombines PCR amplification and DNA detection into one process. TheLightCycler can use one of two processes for detection. The firstprocess relies upon PCR and hybridization. The second process reliesupon PCR and dye and melting curve analysis.

The development of reliable methods for detecting and sequencing nucleicacids is critical to the diagnosis of genetic, bacterial and viraldiseases. Because of the importance of health care and diseaseprevention, a need exists for quicker and cheaper methods of identifyingnucleic acids.

SUMMARY OF THE INVENTION

A method is provided for preparing a test sample for detecting apredetermined target nucleic acid. The method includes the steps ofproviding a test probe comprising an oligonucleotide attached to ananoparticle and providing a hybridization unit containing the testsample and the test probe, wherein said hybridization unit furtherincludes a target sample substrate and a distribution manifold coupledto a first side of the substrate. The method further includes the stepsof clamping a processing fluids manifold to the distribution manifold ofthe hybridization unit, denaturing the test sample and preparing thetest sample for detecting the predetermined target nucleic acid bypumping a plurality of processing fluids between the processing fluidssource manifold and distribution manifold to hybridize the test probeand predetermined target nucleic acid to the target sample substrate, towash the hybridized sample and to amplify a detectable parameter of thehybridized sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a nucleic acid testing system in accordance with anillustrated embodiment of the invention;

FIG. 2 depicts a hybridization unit that may be used with the system ofFIG. 1;

FIG. 3 depicts a manifold that may be used with the hybridization unitof FIG. 2;

FIG. 4 depicts a gasket that may be used with the hybridization unit ofFIG. 2;

FIG. 5 is a schematic of controls that may be used to control the sampleprocessing unit of FIG. 1;

FIG. 6 depicts preparatory steps that may be used in conjunction withthe sample processing system of FIG. 1;

FIG. 7 depicts preparatory steps that may be used in conjunction withthe hybridization unit of FIG. 2;

FIG. 8 depicts loading steps that may occur when the hybridization unitof FIG. 2 is loaded into the sample processing system of FIG. 1;

FIG. 9 depicts operation of the heating/cooling unit of FIG. 8;

FIG. 10 depicts fluid flows in the hybridization unit of FIG. 2;

FIG. 11 depicts a wash cycle that may be used with the sample processingsystem of FIG. 1;

FIG. 12 depicts an amplification step that may be used with the sampleprocessing system of FIG. 1;

FIG. 13 depicts an amplification stop step that may be used with thesample processing system of FIG. 1;

FIG. 14 depicts a flushing step that may be used with the sampleprocessing system of FIG. 1;

FIG. 15 depicts disassembly of the hybridization unit and reading of thesubstrate within the optical reader of FIG. 1;

FIG. 16 is a flow chart of method steps that may be followed by thesample processing system of FIG. 1;

FIG. 17 is a flow chart of a process control application;

FIG. 18 depicts a distribution manifold that may be used with the systemof FIG. 1 under an alternative embodiment of the invention;

FIG. 19 depicts an underside of the distribution manifold of FIG. 18;

FIG. 20 depicts a gasket that may be used with the distribution manifoldof FIG. 18;

FIG. 21 depicts a fluid flow schematic for sample processing under analternate embodiment of the invention.

DETAILED DESCRIPTION OF AN ILLUSTRATED EMBODIMENT

FIG. 1 is a perspective view of a nucleic acid detection system 10,shown generally in accordance with an illustrated embodiment of theinvention. The processing system 10 may be used for the detection of anyof a number of predetermined target nucleic acids. In fact, any type ofnucleic acid may be detected, and the methods may be used for thediagnosis of disease and in sequencing of nucleic acids. Examples ofnucleic acids that can be detected by the methods of the inventioninclude genes (e.g., a gene associated with a particular disease), viralRNA and DNA, bacterial DNA, fungal DNA, cDNA, mRNA, RNA and DNAfragments, oligonucleotides, synthetic oligonucleotides, modifiedoligonucleotides, single-stranded and double-stranded nucleic acids,natural and synthetic nucleic acids, etc. Examples of the uses of themethods of detecting nucleic acids include: the diagnosis and/ormonitoring of viral diseases (e.g., human immunodeficiency virus,hepatitis viruses, herpes viruses, cytomegalovirus, and Epstein-Barrvirus), bacterial diseases (e.g., tuberculosis, Lyme disease, H. pylori,Escherichia coli infections, Legionella infections Mycoplasmainfections, Salmonella infections), sexually transmitted diseases (e.g.,gonorrhea), inherited disorders (e.g., cystic fibrosis, Duchene musculardystrophy, phenylketonuria, sickle cell anemia), and cancers (e.g.,genes associated with the development of cancer); in forensics; in DNAsequencing; for paternity testing; for cell line authentication; formonitoring gene therapy; and for many other purposes.

Included within the system 10 may be a sample processing system 12 andan optical reader 14 for reading samples automatically prepared by thesample processing system 12. The optical reader 14 may be a modelVerigene® ID made by Nanosphere, Inc. of Northbrook, Ill.

The sample processing system 12 may include a controller 300 and anumber of functionally distinct elements used for storage and handlingof processing solutions and samples. For example, the processing system12 may include one or more removable hybridization units 20. Thehybridization unit 20 may be used by the processing system 12 as aprocessing vessel for detecting the predetermined target nucleicacid(s).

The detection system 10 may also require a number of processingsolutions for preparing the nucleic acids for detection. For example,the processing system 12 may require one or more probes 22 and ahybridization buffer fluid (solution) 24. In addition, a processingfluids package 18 may be provided that includes a wash solution, sterilewater, one or more amplifying solutions (e.g., silver part A, silverpart B, etc.) and a stop solution.

The hybridization unit 20 (FIG. 2) may include at least threefunctionally separate portions. A target sample substrate 42 ofoptically transparent glass may be provided as a base for processing thepredetermined nucleic acid. A distribution manifold 44 may be providedthat contacts the substrate and that, together with the substrate 42 anda silicone gasket 58, define the chambers and passageways that allowflow of processing solutions through the hybridization unit 20. Finally,a base 40 is provided that supports the substrate 42.

The manifold 44 may be provided with a flange 43, 45 on opposing sidesthat each contain a set of apertures 56 that resiliently engages acomplementary set of pegs 54 on the base. The pegs 54 may be providedwith a taper on the engagement side to allow the flange to resilientlyexpand over and allow the apertures 56 to engage the pegs 54. Thesilicone gasket 58 (provided on the engagement side of the manifold 44)allows the manifold to resiliently engage with the substrate 42 anddefine a seal around a periphery of chambers and passageways of thehybridization unit 20.

FIG. 3 depicts a simplified view of the manifold 44. FIG. 4 depicts thesilicone gasket 58.

As shown in FIGS. 3 and 4, each hybridization unit 20 may include foursample processing areas 100, 102, 104, 106 (FIG. 4). Each processingarea 100, 102, 104, 106 may include a hybridization zone 140, 142, 144,146 (FIG. 4), an associated sample well 108, 110, 112, 114 (FIG. 3),three processing ports 116, 118, 120; 122, 124, 126; 128, 130, 132; 134,136, 138 (FIG. 3) associated with each respective hybridization zone140, 142, 144, 146 (FIG. 4) and interconnecting passageways (showndisposed in the gasket in FIG. 4).

FIG. 4 shows a range of gasket depths that may be used in conjunctionwith sample processing. It may be noted that the varying depths may beused to minimize flow resistance in the channels while maximizing fluidmixing and interaction among the hybridizing elements within thehybridization chamber 140, 142, 144, 146.

In preparation for testing for a particular nucleic acid, a firstoligonucleotide or first group of oligonucleotides 46, 48, 50, 52 (FIG.2) with a first predetermined genetic sequence may be disposed on thesubstrate 42 (FIG. 2) within each of the hybridization zones 140, 142,144, 146 (FIG. 4). The first oligonucleotides 46, 48, 50, 52 (FIG. 2)may have a genetic sequence that is complementary to a first portion ofthe genetic sequence of the predetermined target nucleic acid.

The probes 22 (FIG. 1) may be constructed of nanoparticles with one ormore strands of second oligonucleotides of a second predeterminedgenetic sequence attached to the nanoparticles. Nanoparticles useful inthe practice of the invention may include metal (e.g., gold, silver,copper, and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSecoated with ZnS) and magnetic (e.g., ferromagnetite) colloidalmaterials. Other nanoparticles useful in the practice of the inventioninclude ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe, In₂S₃,Cd₃P₂, Cd₃As₂, InAs, and GaAs. The size of the nanoparticles ispreferably from about 5 nm to about 150 nm (mean diameter), morepreferably from about 5 to about 50 nm, most preferably from about 10 toabout 30 nm.

The nanoparticles, the second oligonucleotides or both arefunctionalized in order to attach the oligonucleotides to thenanoparticles. Such methods are known in the art. For instance,oligonucleotides functionalized with alkanethiols at their 3′-termini or5′-termini readily attach to gold nanoparticles.

The second oligonucleotides may have a sequence that is complementary toa second portion of the genetic sequence of the predetermined targetnucleic acid. Preparation of the first and second oligonucleotides andattachment to the respective particles and substrate may be accomplishedgenerally as described in U.S. Pat. No. 6,417,340 assigned to theassignee of the present invention and incorporated herein by reference.

In general, the test probe and test sample (that may or may not containthe predetermined target nucleic acid) and a hybridization fluid may bemixed in a sample well. The mixture may be denatured and passed throughthe hybridization chamber. Denaturing may be accomplished using anyknown process (e.g., heat, chemical, etc.).

Within the hybridization chamber, the test probe and predeterminednucleic acid may hybridize with the first oligonucleotide. The opticalcharacteristics of the hybridized materials may be enhanced (e.g.,plating a silver solution to the nanoparticles of the hybrid). Afterenhancement, any hybridized materials may be detected optically withinthe optical reader 14 (FIG. 1). In this case, the plating of the silversolution to the gold nanoparticles of the hybrid amplifies the opticalreflectivity of the hybrid. The optical reflectivity may then becompared with a threshold value to confirm the presence of the targetnucleic acid.

Alternatively, the detectable parameter may be resistance. In this case,the silver plated to the gold nanoparticle within the hybrid amplifies acurrent path through the hybrid. The resistance may then be comparedwith a threshold value to confirm the presence of the target nucleicacid.

Turning now to operation of the sample processing system 12 (FIG. 1), anexplanation will now be provided of the controller 300 and theinteraction of the controller 300 with the hybridization unit 20. Inthis regard, FIG. 5 depicts the controller 300 and various actuatingelements used by the sample processing system 12 in processing sampleswithin the hybridization unit 20.

Along the right side of FIG. 5 is shown a set of ports labeled “SAMPLEWELL, B, A, C”. The reference “SAMPLE WELL” may be used to refer tolarge ports 108, 110, 112, 114 in FIG. 3. Similarly, port B may be usedto refer to smaller ports 116, 126, 128, 138, port A may be used torefer to ports 118, 124, 130, 136 and port C may be used to refer toports 120, 122, 132, 134.

Processing of samples in sample processing areas 100, 102, 104, 106(FIG. 4) may be assumed to be substantially identical. It should benoted in this regard that while the processing may be substantiallyidentical for each sample processing area 100, 102, 104, 106, the targetnucleic acid that is detected may be different within each of the fourareas 100, 102, 104, 106.

As shown in FIG. 5, the sample processing system 12 may include a numberof pumps 302, 304, 306, 308, 310, 312 and a vacuum source 314. While anyform of pump 302, 304, 306, 308, 310, 312 may be used, it iscontemplated that a positive displacement pump such as a syringe pumpmay be used for reasons that will become apparent from the descriptionbelow.

The syringe pumps may include a syringe body and a linear actuator. Thelinear actuator may be programmed by the controller 300 to fill andempty at precisely controlled rates.

The routing of fluids to and from the pumps 302, 304, 306, 308, 310, 312may be controlled by a number of multiport valves 316, 318, 320, 322,324, 326. While any number of ports may be used, it is believed that thefour-port valves 316, 318, 320, 322, 324, 326 shown in FIG. 5 areparticularly well adapted to the purpose described below.

In this regard, the valves 316, 318, 320, 322, 324, 326 may have portslabeled 1-4. A spool within the valves 316, 318, 320, 322, 324, 326 mayallow any two opposing ports to be connected together (e.g., port 1 maybe connected to port 3 or port 2 may be connected to port 4).

When used with syringe pumps 302, 304, 306, 308, 310, 312, multiportvalves 316, 318, 320, 322, 324, 326 allow a precise amount of a selectedfluid to be transferred at each stage of processing. For example, withports 1 and 3 of valve 322 connected (as shown in FIG. 5), the syringepump 308 may withdraw a precise amount of water from the water container334 on a fill portion of the pump cycle. The multiport valve 322 maythen be actuated to connect ports 2 and 4. The water previously drawninto the syringe pump 308 may now be discharged through port 4 of valve322 and into port A of the hybridization unit 20.

It may be noted that in some applications, the valves 316, 318, 320,322, 324, 326 and containers 328, 330, 332, 334, 336, 338 may not beneeded. For example, the valves and containers would not be needed incases where the total flow for each function is less than the capacityof the syringe pump 302, 304, 306, 308, 310. In these cases, the syringepump may simply be replaced after each testing procedure or aftermultiples of each testing procedure.

FIG. 21 illustrates fluid flow for sample processing under an even morepreferred embodiment of the invention. In the example of FIG. 21,fluidic control is maintained without the use of valves by utilizingpumps on the inlet and outlet ports to route fluids down a specifiedpath.

By introducing fluids via pump 1 and only withdrawing fluids via pump 2,the fluid can be routed through hybridization chamber and flow path A.Fluids can also be routed down multiple paths in parallel by actuatingthe control pumps (2, 3, 4 or 5) for that fluid path. Parallel fluidprocessing may be useful to reduce processing time when high tolerancepumping is not required, such as during washing and rinsing steps.

Other additional pumps on the inlet side (not shown in FIG. 21) willprovide additional fluids. A system such as that shown in FIG. 21 with 8inlet pumps for 8 specific fluids can perform a variety of nucleic acidtests. The type of tests can be selected by the insertion of variousfluids into the flow paths from the 8 pump chambers.

Access to fluids inserted into the sample well by the user isaccomplished by pulling on the outlet pump(s) only. The sample well isdesigned to easily collapse and block flow so that the target samplewill flow preferentially only out of the specific sample well for thespecific flow path and outlet pump desired.

FIGS. 6-16 show process steps that may be used in detecting thepredetermined nucleic acid. For example, Frame #1 of FIG. 6 shows thepreliminary step of providing a reagent cartridge 18 and a wastecontainer 16. Frame #2 shows the loading of the cartridges 16, 18 intothe sample processing system 12. Frame #3 shows the closing of the doorand references the fact that closing the door causes a set of connectionfittings to puncture the seals on the reagent and waste containers.Alternatively, or in addition, closing the door provides a signal to thecontroller which then controls linear actuators to engage the pumpswhich provides fluid(s) for processing. A bar code reader 340 (FIG. 8)may be provided to read a bar code on the reagent cartridge toautomatically verify that the correct reagent cartridge has beeninserted.

FIG. 7 shows preparation of the hybridization unit 20. Frame #1 shows auser opening a set of lids covering the four sample wells 108, 110, 112,114 (FIG. 3). The user than pipettes a hybridization buffer into thefour wells 108, 110, 112, 114 as shown in Frame #2. The user thenpipettes the probe 24 into the four wells 108, 110, 112, 114, as shownin Frame #3. As a fourth step, the user pipettes a sample that maycontain the predetermined target nucleic acid into the well 108, 110,112, 114 as shown in Frame #4. Finally, the user closes the lids on thewells 108, 110, 112, 114 as shown in Frame #5. Alternatively, the usermay provide only the predetermined target nucleic acid into the samplewell or a combination of the predetermined target nucleic acid andhybridization buffer or predetermined target nucleic acid and probe intothe sample well.

FIG. 8 shows preparation and loading of the hybridization unit 20 intothe sample processing system 12. As a first step, shown in Frame #1 ofFIG. 8, the user may use a barcode reader 340 to identify thehybridization unit 20 to the system 12. Alternatively, the bar codereader may be embedded inside the loading door of the system and the barcode may be read when the hybridization unit is loaded into the system.

To load the hybridization unit 20, the user may open a door on thesample processing system 12. A spring-loaded receptacle that catchesfluid from a fluid manifold 72 of the processing system 12 is foundextended to a fully forward position as shown in Frame 2 of FIG. 8. Theuser then pushes the hybridization unit 20 into the sample processingsystem 12 as shown in Frame #3 and closes the door (Frame #4).

Activation of the sample processing system 12 may occur by closure ofthe door or by activating a START button 342 (FIG. 1). In either case,activation of the system 12 causes the hybridization unit 20 to beraised into contact with a processing fluids manifold 72 and aheating/cooling block 60 to be raised into contact with thehybridization unit 20 (FIG. 8, Frame #5). The raising of thehybridization unit 20 and heating/cooling block 60 may be accomplishedby a simple mechanical linkage connected to the door or through a linearactuator coupled to an elevator assembly.

The raising of the hybridization unit 20 creates a fluid-transferconnection between the ports 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138 of the hybridization unit 20 and respective ports ofthe processing fluids manifold 72 and with the pumps 1-7 of FIG. 21 orwith respective valves 316, 318, 320, 322, 324, 326 and with pumps 302,304, 306, 308, 310, 312, 314 of the sample processing system 12 of FIG.5. Similarly, the raising of the heating/cooling block 60 causes athermal transfer connection between the hybridization unit 20 and theheating/cooling block 60.

FIG. 9 depicts a preliminary processing step 400 (FIG. 16) performed bythe sample processing system 12. As shown, a first heating element 62and a second heating element 70 of the heating/cooling block 60 connectto and heat the sample wells 108, 110, 112, 114 to a temperature fordenaturing the samples (e.g., 95° C.) of the predetermined targetnucleic acid. As used herein, denature means to cause the tertiarystructure of the nucleic acid to unfold.

A first cooling element 64 and a second cooling element 68 function tocool the denatured samples as they are transferred from the sample wells108, 110, 112, 114 to hybridization chambers 140, 142, 144, 146. A thirdheating element 66 is located adjacent the hybridization chambers 140,142, 144, 146 to heat the samples to a specified temperature forhybridization (e.g., 40° C.).

Frame #1 of FIG. 10 depicts the heating of the samples in the samplewells 108, 110, 112, 114 to the denaturing temperature (e.g., 95° C.).Frame #2 of FIG. 10 depicts loading 402 (FIG. 16) of the samples bytransferring the samples through the chill zone into the hybridizationchambers 140, 142, 144, 146. Transfer of the samples from the samplewells 108, 110, 112, 114 may be accomplished by activating the wastepump 312 with the waste valve 324 in the position shown in FIG. 5. Thetransfer of the samples across the chill zone may be accomplished by thecontroller 300 choosing a relatively slow rate of fluid transfer (e.g.,1 cc/min) as the pump 312 pulls fluid from port C to ensure propercooling of the samples as they pass over the chill zone.

It may be noted that to load the sample into the hybridization zone 140,142, 144, 146, the controller 300 may retrieve and execute a set ofvalve and motor control parameters (instructions) 346 (FIG. 5) frommemory for controlling a linear actuator of the pump 312. The parameters346 may include a motor identifier 348, a direction 350, a speed 352, atime 354 and a valve position 356.

If the linear actuator has its own controller, then the direction 350,speed 352 and time may be simply downloaded to the controller forexecution. If the controller is provided through the use of specialpurpose programs within the controller 300, then execution of theinstructions may be provided from within the controller 300.

It should be noted that (before loading of the samples) thehybridization chambers 140, 142, 144 may initially have been filled withair. As such, the fluid pulled from port C would be air. The withdrawalof air from port C pulls the samples from the sample wells 108, 110,112, 114 into the hybridization chambers 140, 142, 144, 146.

As a final step in the process of loading the sample, the controller 300may reset the waste pump 310. Resetting the waste pump 310 may meanretrieving a set of instructions 358 (FIG. 5) from memory. Theinstructions 358 may contain an instruction 368 that causes the wastevalve 326 forms a connection between ports 2 and 4. The instructions 358may also contain a motor identifier 360, a direction 362, a speed 364and a time 366 necessary to cause the waste pump 310 to move to a fullydischarged position.

It may be noted that the instructions for loading the sample and forresetting the waste pump 310 and for performing the other process stepsdescribed herein may be accomplished by a process control applicationdepicted in FIGS. 16 and 17. FIG. 16 may be used to depict the overallfunctionality of the control application and FIG. 17 may be used todepict the activity performed within the individual blocks of FIG. 16.

With respect to execution of the control application, activation of theSTART button 342 or closing the door brings the hybridization unit 20into contact with the manifold 72 and heating/cooling block 60.Activation may also start a timer within the controller 300 to detectcompletion of the denaturization process 400. From the denaturizationprocess 400, the control application proceeds to the load sample process402. As a first step of the load sample process 402, the application 500loads and executes the load sample file 346. As a second step, theapplication 500 loads and executes the reset pump files 358. In eachcase, the application 500 positions the valves, loads actuatorpositioning parameters and executes the positioning parameters. Onceeach process is complete, the application 500 advances to the nextprocess step.

Frame #3 of FIG. 10 depicts hybridization of the sample and probe withthe oligonucleotide strands within hybridization chamber 140, 142, 144,146. In this case, the controller 300 functions to shuttle 404 thepartially hybridized sample and probe back and forth across thehybridization chamber 140, 142, 144, 146.

To shuttle the partially hybridized sample back and forth across thehybridization chamber 140, 142, 144, 146, the application 500 retrievesand execute a set of instructions 370, 372 that activate the wash pump310 and waste pump 312 to move in opposite directions. In this case, theinstructions 370, 372 would cause the wash valve 324 to form aconnection between ports 2 and 4 and the waste valve 326 to form aconnection between ports 1 and 3. The shuttle forward instruction 370may cause the wash pump 310 to move a predefined distance towards anempty position and the waste pump 312 to move a predefined distancetowards a filled position. When the wash pump 310 and waste pump 312reach the predefined distance, the application 500 would execute theshuttle reverse instructions 372. The shuttle reverse instruction 372may cause the wash pump 310 to move a similar distance towards a fullposition and the waste pump 312 to move a similar towards an emptyposition. When the predetermined distances are reached, the application500 may again execute the shuttle forward instructions 370.

Each time the application 500 executes the shuttle forward instructions370, a counter 374 is incremented 406. After each increment, the valuewithin the counter 374 may be compared 408 within a comparator 376 witha shuttle cycle limit value that terminates the shuttling process aftera predefined number of cycles.

Since the pumps 310, 312 would initially contain air, the reciprocalaction of the pumps 310, 312 would simply push the sample into and outof the passageways on either end of the hybridization chamber 140, 142,144, 146 with very little if any of the partially hybridized sampleentering either pump 310, 312. Shuttling of the partially hybridizedsample across the hybridization zones 140, 142, 144, 146 may continuefor a time period determined by the identity and type of the sample(e.g., 10-60 minutes).

Following hybridization of the sample and probe with the oligonucleotidestrands within the hybridization chamber 140, 142, 144, 146, thehybridized materials may be washed 410 as shown in Frames #1 and #2 ofFIG. 11. To wash the hybridized materials, the controller 300 mayexecute a set of wash instructions 378 that may concurrently activatethe wash pump 310 and the waste pump 312. As a first step, theinstructions 378 may cause the wash valve 324 to form a connectionbetween ports 1 and 3. The wash pump 310 may then be activated to drawwater from a wash container 336.

Once the syringe pump 310 is full, the instructions 378 may cause thevalve 324 to form a connection between ports 2 and 4. The waste valve326 may also be moved to form a connection between ports 1 and 3. Thewash pump 310 and waste pump 312 may be simultaneously activated tooperate at the same rate. The wash pump 310 functions to push water intoport A and the waste pump 312 functions to pull fluids out of port C.

When the syringe of the wash pump 310 reaches its empty position, thewaste pump 312 would reach its full position. At this stage, the washvalve 324 may move to form a connection between ports 1 and 3 and thewaste valve 326 may move to form a connection between ports 2 and 4. Thewash pump 310 and waste pump 312 may again be activated. In this case,the wash pump 310 now refills from the wash container 336 and the wastepump 312 now discharges into the waste container 338. The fill and emptyprocess may repeat for the number of cycles necessary to flush anyun-hybridized materials from the hybridization unit 20. A counter may beincremented after each fill and empty cycle and a value within thecounter may be compared with a cycle limit within a comparator todetermine completion of the wash cycle.

Once the hybridized materials have been washed, a detectable parameterof the hybridized materials may be amplified to allow detection of thehybridization. The detectable parameter may be any measurable quantitythat indicates the presence or absence of the hybridized materials.Under illustrated embodiments the optical or conductive properties ofthe hybridized materials may be amplified 412 for purposes of detection.Amplification, in this case occurs by plating a silver solution onto thenanoparticles of the hybrid.

Amplification may occur by passing a silver A solution and a silver Bsolution through the hybridization chamber 140, 142, 144, 146. To passthe silver A solution and silver B solutions through the hybridizationchamber, the controller 300 may execute a set of instructions 380 thatcauses silver A valve 320 and the silver B valve 316 to form aconnection between ports 1 and 3. The silver A pump 306 and silver Bpump 302 may then be activated by the instructions 380 to draw thesilver A solution from the silver A container 332 into the silver A pump306 and the silver B solution from the silver B container 328 into thesilver B pump 302.

The silver A valve 320 and the silver B valve 316 may then be instructedto form a connection between ports 2 and 4. The waste valve 326 may beinstructed to form a connection between ports 2 and 4. The instructions380 may specify a discharge rate for silver A pump 306 and the silver Bpump 302 and the controller 300 may activate the pumps 306, 302 todischarge at those rates. The silver A pump 306 may discharge into portA and the silver B pump 302 may discharge into port B. The instructions380 may also specify an intake rate for the waste pump 312 equal to anoutput of the silver A pump 306 and silver B pump 302 and the controller300 may activate the waste pump 312 to withdraw fluid from the port C atthe selected rate. Once the silver A pump 306 and the silver B pump 302have discharged their materials into the respective ports and the wastepump 312 has been filled with fluid withdrawn from port C, the valves316, 320, 326 may again be moved under control of the instructions 380.The silver A valve 320 and the silver B valve 316 may be positioned toagain fill the silver A pump 306 and silver B pump 302 with silversolutions. The waste valve 326 may be positioned to discharge withdrawnmaterials into the waste container 338. The fill and empty steps may berepeated by the number of cycles necessary for sufficient amplificationof the hybridized materials again under the control of a counter andcomparator based upon a cycle limit value.

Once the amplification step has been completed, a stop solution may bepassed through the hybridization chambers 140, 142, 144, 146 as shown inFIG. 13 to stop amplification 414. In this regard, a set of stopinstruction may be executed by the controller 300 to position the stopvalve 318 with a connection between ports 1 and 3. The stop pump 304 maybe activated to fill the pump 304 from the stop solution container 330.The controller 300 under control of the instructions 382 may then movethe stop valve to form a connection between ports 2 and 4 and the wastevalve to form a connection between ports 1 and 3. The controller 300 maythen select a discharge rate for the stop pump 304 and activate the stoppump 304. The controller 300 may select the same withdrawal rate for thewaste pump 312 and simultaneously activate the waste pump 312 to pullthe stop solution through the hybridization chamber 140, 142, 144, 146.The valves 318, 326 may be repositioned to refill the stop pump 304 andempty the waste pump 312 and the process may be repeated.

Under an even more preferred embodiment, the pumps would never berefilled. In this case, the pump bodies are integrated into a reagentcartridge that is simply replaced when empty.

Once the stop solution has been passed through the hybridization chamber140, 142, 144, 146, the hybridization chamber 140, 142, 144, 146 may beflushed 416 with dd water and vacuumed to remove residual fluid as shownin FIG. 14.

To flush the hybridization chambers 140, 142, 144, 146, the controller300 operating under flush instructions 384 may move the flush valve 322to form a connection between ports 1 and 3 and activate the flush pump308 to fill with water from the water container 334. The controller 300may then reposition the flush valve 322 to allow the flush pump 308 todischarge into port A and reposition the waste valve 326 to withdrawfluid from port C. Once the flush pump 308 is empty, the valves 322, 326may be repositioned to refill the flush pump 308 and empty the wastepump 312 and the process may be repeated.

Once flushing is complete, the controller 300 operating under control ofinstructions 384 may activate the vacuum 314. The vacuum 314 may pullany remaining fluids out of the hybridization unit 20 by displacing thefluids with air pulled in through the respective sample wells 108, 110,112, 114.

Once any remaining fluids have been removed, the sample processing unit12 may unlock as shown in FIG. 15 and the hybridization units 20 may beremoved. The substrate 58 may be removed from the hybridization unit 20and placed in the optical reader 14 where the optical characteristics ofthe hybridized sample may be read.

In another illustrated embodiment of the invention (shown in FIGS.18-20), the distribution manifold 44 shown on the hybridization unit 20of FIG. 2 is replaced with a distribution 600 (shown as a completehybridization unit 20 in FIG. 18). FIG. 19 shows a reverse view of themanifold 600. FIG. 20 shows a gasket 700 that may be used with themanifold 600 of FIGS. 18 and 19.

As with the manifold 44 of FIG. 2, the distribution manifold 600 of FIG.18 has sample wells 602, 604, 606, 608 in opposing corners. Thisdistribution manifold 600 has four waste ports 610, 612, 614, 616associated with a respective hybridization zone 708, 706, 704, 702 (FIG.20). Also shown in FIG. 19 is a common fill port. The manifold 600 ofFIGS. 18, 19 and 20 is believed to be particularly well adapted for usewith the system of FIG. 21.

FIG. 19 shows an underside of the distribution manifold 600 of FIG. 18.As shown, each of the ports 602, 604, 606, 608, 610, 612, 614, 616, 618of FIG. 18 has a corresponding feedthrough 602, 604, 606, 608, 610, 612,614, 616, 618. It should also be noted that the fill port 618 has achannel 620 disposed on a surface of the distribution manifold 600 thatterminates at four feedthrough points 622, 624, 626, 628.

Turning now to the gasket 700 (FIG. 20), it may be noted that the gasket700 defines the hybridization chambers 702, 704, 706, 708 and a numberof connecting channels. For example, the first hybridization chamber 702has a connecting channel that connects the sample well 606, feedthrough628 and the first end of the hybridization chamber 702. The firsthybridization chamber 702 also a connecting channel that connects asecond end of the hybridization chamber 702 to waste port 616.

The second hybridization chamber 704 has a connecting channel thatconnects the sample well 608, feedthrough 622 and the first end of thehybridization chamber 704. The second hybridization chamber 704 also aconnecting channel that connects a second end of the hybridizationchamber 704 to process port 614.

The third hybridization chamber 706 has a connecting channel thatconnects the sample well 602, feedthrough 624 and the first end of thehybridization chamber 706. The third hybridization chamber 706 also aconnecting channel that connects a second end of the hybridizationchamber 706 to process port 612.

Similarly, the fourth hybridization chamber 708 has a connecting channelthat connects the sample well 604, feedthrough 626 and the first end ofthe hybridization chamber 708. The second hybridization chamber 708 alsoa connecting channel that connects a second end of the hybridizationchamber 708 to process port 610.

It should be noted that the fluid manifold 72 and pump connections withthe processing unit 12 may also be changed to accommodate thedistribution manifold 600. It may be noted in this regard that portconnections A and B in FIG. 5 would be combined and connected to therespective process port 610, 612, 614, 616. The waste port 618 in FIG.18 would correspond to port C in FIG. 5. In other regards, ahybridization unit 20 using the distribution manifold 600 would operatesubstantially the same as described above.

In another illustrated embodiment of the invention, the manifold 72 maybe provided with a connection to replaceable cartridges for thehybridization buffer and/or probes. Under this embodiment, the userwould simply add the target sample to the test wells and insert thehybridization unit 20 into the sample processing system 12. The system12 would add any missing elements to the sample wells.

A specific embodiment of method and apparatus for processing nucleicacid samples has been described for the purpose of illustrating themanner in which the invention is made and used. It should be understoodthat the implementation of other variations and modifications of theinvention and its various aspects will be apparent to one skilled in theart, and that the invention is not limited by the specific embodimentsdescribed. Therefore, it is contemplated to cover the present inventionand any and all modifications, variations, or equivalents that fallwithin the true spirit and scope of the basic underlying principlesdisclosed and claimed herein.

1. A method of preparing a test sample for purposes of detecting apredetermined target nucleic acid, such method comprising the steps of:(a) providing a test probe comprising an oligonucleotide attached to ananoparticle; (b) providing a hybridization unit containing the testsample and the test probe, wherein said hybridization unit furthercomprises a target sample substrate and a distribution manifold coupledto a first side of the substrate; (c) clamping a processing fluidsmanifold to the distribution manifold of the hybridization unit; (d)denaturing the test sample; and (e) preparing the test sample fordetecting the predetermined target nucleic acid by pumping a pluralityof processing fluids between the processing fluids source manifold anddistribution manifold to hybridize the test probe and predeterminedtarget nucleic acid to the target sample substrate, to wash thehybridized sample and to amplify a detectable parameter of thehybridized sample.
 2. The method of preparing the test sample as inclaim 1 further comprising disposing a hybridization solution into asample well of the hybridization unit.
 3. The method of preparing thetest sample as in claim 2 further comprising disposing the test probeinto the sample well.
 4. The method of preparing the test sample as inclaim 1 further comprising disposing the test sample in the sample well.5. The method of preparing the test sample as in claim 4 wherein thestep of denaturing the test sample further comprises heating the samplewell.
 6. The method of preparing the test sample as in claim 5 furthercomprising disposing an oligonucleotide having a sequence complementaryto a first portion of a genetic sequence of the predetermined targetnucleic acid within a hybridization zone of the hybridization unit. 7.The method of preparing the test sample as in claim 6 wherein the stepof disposing the oligonucleotide within a hybridization zone of thehybridization unit further comprises connecting the oligonucleotide tothe target sample substrate.
 8. The method of preparing the test sampleas in claim 7 further comprising defining the oligonucleotide attachedto the nanoparticle as having a genetic sequence complementary to asecond portion of the genetic sequence of the predetermined targetnucleic acid.
 9. The method of preparing the test sample as in claim 8further comprising drawing a content of the sample well from the samplewell into the hybridization zone using a fluid coupled through a firstport of the processing fluids manifold.
 10. The method of preparing thetest sample as in claim 9 further comprising chilling the content of thesample well as the content is drawn into the hybridization zone.
 11. Themethod of preparing the test sample as in claim 9 further comprisinghybridizing the probe and predetermined nucleic acid with theoligonucleotide connected to the target sample substrate by shuttlingthe content of the well through the hybridization zone a predeterminednumber of times.
 12. The method of preparing the test sample as in claim11 further comprising flushing the hybridized probe, predeterminednucleic acid and first oligonucleotide by introducing wash fluid througha second port and discharging wash fluid through the first port.
 13. Themethod of preparing the test sample as in claim 12 further comprisingamplifying optical characteristics of the hybridized probe,predetermined nucleic acid and first oligonucleotide by introducing aplating solution through a second port and discharging spent platingsolution through the first port.
 14. The method of preparing the testsample as in claim 13 further comprising defining the plating solutionas being a silver solution.
 15. A method of preparing a plurality oftest samples for purposes of detecting predetermined target nucleicacids, such method comprising the steps of: (a) providing ahybridization unit containing the plurality of test samples, saidhybridization unit further comprising a target samples substrate and adistribution manifold coupled to a first side of the substrate saidtarget samples; (b) clamping a processing fluids manifold to thedistribution manifold of the hybridization unit; (c) denaturing the testsamples; and (d) preparing the plurality of test samples for detectingthe predetermined nucleic acids by pumping a plurality of processingfluids between the processing fluids source manifold and distributionmanifold to hybridize the predetermined target nucleic acid to thetarget samples substrate, to wash the hybridized samples and to amplifya detectable parameter of the hybridized samples.
 16. The method ofpreparing the test sample as in claim 15 further comprising disposingthe plurality of test samples in a plurality of respective sample wells.17. The method of preparing the test sample as in claim 16 wherein thestep of denaturing the test samples further comprises heating a samplewell of the plurality of respective sample wells.
 18. The method ofpreparing the test sample as in claim 17 further comprising disposing aliquid test probe into a first well of the plurality of sample wells.19. The method of preparing the test sample as in claim 18 furthercomprising disposing an first oligonucleotide having a sequencecomplementary to a first portion of a sequence of a nucleic acid of thepredetermined nucleic acids within a hybridization zone of thehybridization unit.
 20. The method of preparing the test sample as inclaim 19 wherein the step of disposing the first oligonucleotide withina hybridization zone of the hybridization unit further comprisesconnecting an end of the oligonucleotide to the target samplessubstrate.
 21. The method of preparing the test sample as in claim 20wherein the test probe further comprises a nanoparticle and a secondoligonucleotide disposed on a surface of the nanoparticle, said secondoligonucleotide having a sequence complementary to a second portion ofthe sequence of the nucleic acid of the predetermined nucleic acids. 22.The method of preparing the test sample as in claim 21 furthercomprising drawing a content of a well of the plurality of wells fromthe well into the hybridization zone using a fluid coupled through afirst port of the processing fluids manifold.
 23. The method ofpreparing the test sample as in claim 22 further comprising cooling thecontent of the sample well of the sample wells as the content is drawninto the hybridization zone.
 24. The method of preparing the test sampleas in claim 22 further comprising hybridizing the probe andpredetermined nucleic acid with the first oligonucleotide by shuttlingthe content of the well through the hybridization zone a predeterminednumber of times to mix the probe and predetermined nucleic acid with thefirst oligonucleotide.
 25. The method of preparing the test sample as inclaim 24 further comprising flushing the hybridized probe, predeterminednucleic acid and first oligonucleotide by introducing wash fluid througha second port and discharging wash fluid through the first port.
 26. Themethod of preparing the test sample as in claim 25 further comprisingamplifying optical characteristics of the hybridized probe,predetermined nucleic acid and first oligonucleotide by introducing aplating solution through a second port and discharging spent platingsolution through the first port.
 27. The method of preparing the testsample as in claim 26 further comprising defining the plating solutionas being a silver solution.